Real‐time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no‐wash, no‐lyse kinetic method

Balaguer, Susana; Díaz, L.; Gomes, Ana Julia Pereira Santinho; Herrera, Guadalupe; O’Connor, José-Enrique; Urios, Amparo; Felipo, Vicente; Montoliú, Carmina

doi:10.1002/cyto.b.21237

Cited by 14 publications

(8 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To identify the mechanism of fungal killing by PMN-DCs, we quantified ROS and NO production during blastomycosis. We stained leukocytes ex vivo with dihydrorhodamine-123 and DAF-FM, fluorescent indicators of ROS and NO respectively [ 41 ]. While similar proportions of PMN-DCs and canonical neutrophils produced ROS, PMN-DCs produced more ROS than canonical neutrophils ( Fig 3D ).…”

Section: Resultsmentioning

confidence: 99%

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

et al. 2018

View full text Add to dashboard Cite

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

show abstract

Section: Resultsmentioning

confidence: 99%

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Since then, publications using kinetic flow-cytometry started to be reported (24)(25)(26)(27)(28). Subsequently, the technique was improved and redefined as "continuous" (29) or "real-time flow cytometry" (30,31). In the platelet research field, kinetic flow cytometry was at first applied to investigations of calcium kinetics fluxes (24,(32)(33)(34)(35)(36) or activation markers (35,37).…”

mentioning

confidence: 99%

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

2020

View full text Add to dashboard Cite

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow‐cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real‐time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo‐3 (Ca2+), ION NaTRIUM Green‐2 (Na+), and ION Potassium Green‐2 (K+). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR‐1 agonist), AYPGKF (PAR‐4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry

show abstract

“…Analysis was performed on a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA) and the Kaluza software package was used to analyze the flow cytometry data. The cytometer settings were performed as in Balaguer et al [28].…”

Section: Methodsmentioning

confidence: 99%

Selective improvement by rifaximin of changes in the immunophenotype in patients who improve minimal hepatic encephalopathy

et al. 2019

Self Cite

View full text Add to dashboard Cite

Background Minimal hepatic encephalopathy (MHE) in cirrhotic patients is associated with specific changes in parameters of the immune system reflecting a more pro-inflammatory environment than in patients without MHE. The aims of this work were to assess the effects of rifaximin treatment of cirrhotic patients with MHE on: (1) MHE; (2) intermediate (CD14 ++ CD16 + ) pro-inflammatory monocytes; (3) expression of early activation marker CD69 in T lymphocytes; (4) autoreactive CD4 + CD28 − T lymphocytes; (5) differentiation of CD4 + T lymphocytes to Th follicular and Th22; (6) serum IgG levels; and (7) levels of some pro-inflammatory cytokines. Methods These parameters were measured by immunophenotyping and cytokine profile analysis in 30 controls without liver disease, 30 cirrhotic patients without MHE and 22 patients with MHE. Patients with MHE were treated with rifaximin and the same parameters were measured at 3 and 6 months of treatment. We assessed if changes in these parameters are different in patients who improve MHE (responders) and those who remain in MHE (non-responders). Results Rifaximin improved MHE in 59% of patients with MHE. In these responder patients rifaximin normalized all alterations in the immune system measured while in non-responders it normalizes only IL-6, CCL20, and differentiation of T lymphocytes to Th22. Non-responder patients do not show increased expression of CD69 before treatment. Conclusions Rifaximin normalizes changes in the immune system in patients who improve MHE but not in non-responders. Some alterations before treatment are different in responders and non-responders. Understanding these differences may identify predictors of the response of MHE to rifaximin.

show abstract

Real‐time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no‐wash, no‐lyse kinetic method

Abstract: This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society.

Cited by 14 publications

References 28 publications

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

Selective improvement by rifaximin of changes in the immunophenotype in patients who improve minimal hepatic encephalopathy

Contact Info

Product

Resources

About